Real-time detection of DNA amplification during polymerase chain reaction (PCR) provides quantitative data for amplified DNA target sequences by relating the number of temperature cycles during thermal cycling needed to reach the concentration threshold (Ct) of the target sequence to the amount of target DNA present at the start of the PCR process. Thus, accurate and reproducible determination of the amount of target sequence present can be effected by detecting accurate Ct values. One aspect to determining accurate Ct values is to relate the fluorescent signal generated during thermal cycling to a passive internal reference dye for normalization of signal across samples and correction of well-to-to well optical variation in high-throughput multiwell assays (see, e.g., Real-time PCR, ed. Julie Logan, Kristin Edwards, Nick Saunders, 2009; Wong, M L and Medrano, J F, Biotechniques, 39:75-85, 2005 and Gehua et al. Can. J. Microbiol., 53:391-397, 2007). Since excitation optics vary between different instrument platforms, the optimal passive reference dye concentration must be matched to a specific real-time thermal cycler.
5- and/or 6-carboxy-X-rhodamine (e.g., available commercially as ROX™) is commonly used as a passive reference dye in a number of instruments. The passive (not interacting with components of a nucleic acid amplification reaction, not participating in 5′-exonuclease-induced fluorescent signal generation (if used), not affecting amplification reaction efficiency, etc.) reference, 5- or 6-carboxy-X-rhodamine dye, provides an internal reference to which the reporter dye signal can be normalized. This signal normalization allows one to correct for fluorescent fluctuations due to changes in concentration or volume. Normalization of the reporter dye signal results in increased data precision and reproducibility among replicate reactions. In some ‘normalization’ calculations the fluorescence emission intensity of the reporter dye or other fluorescent signal indicating amplicon quantity is divided by the fluorescence emission intensity of a passive reference dye (e.g., 5- or 6-carboxy-X-rhodamine dye) to obtain a ratio for the normalized reporter or Rn. The difference between the values of the baseline and the template-containing sample equals the delta (Δ)Rn, indicating the magnitude of signal generated by the sample under those specific PCR conditions. Absolute or relative expression can be determined on the basis of an assigned threshold and the number of cycles needed to cross that threshold (Ct, Cp or Cq).
Due to differences in design of thermal cyclers, some instruments employ a high concentration of 5- or 6-carboxy-X-rhodamine dye for normalization while others employ low concentration. On a high-Rox instrument, such as AB7900, the light source is laser-based with excitation at 480 nm. When the Rox dye, which has maximum absorbance at 580 nm, is used for passive normalization, it is not efficiently excited by the 480 nm laser. To compensate for the low efficiency of excitation of the Rox dye and to generate sufficient signal for proper normalization, very high concentration of the Rox dye has to be used. Thus this instrument is referred to as the “high-Rox” platform. While many other real-time platforms detect signal in different channels, the AB7900 collects signals in different bins, with a set of bins designated for ROX.
On a low-Rox instrument such as AB7500, a broad-spectrum light source is used, which is then divided into 4 excitation wavelength through the use of filters for the corresponding channels. The emission is detected within each designated channel. Therefore, the Rox dye can be maximally excited in the “Rox” channel and generating adequate signal for normalization without requiring high concentration of the Rox dye. Thus, such instrument and alike are referred to as the “low-Rox” platforms. Typically, the Rox concentration used by a “high-Rox” platform is about 10-fold higher than that used by a “low-Rox” platform.